Note: Descriptions are shown in the official language in which they were submitted.
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USE OF A 5-HT6 AGONIST FOR THE TREATMENT AND PREVENTION OF
NEURODEGENERATIVE DISORDERS
BACKGROUND OF THE INVENTION
Glutamate is the predominant neurotransmitter in the central nervous system
and plays and important role in neuroplasticity. Excessive extracellular
levels of
glutamate have been associated with the pathophysiology of both acute
neurodegenerative disorders such as stroke, transient ischemic attack or
spinal/brain
trauma, and chronic neurodegenerative disorders such as epilepsy, Alzheimer's
Disease, amyotrophic lateral sclerosis, Huntington's disease, Parkinson's
Disease,
AIDS dementia and retinal diseases'. Compounds which inhibit the release of
glutamate would be expected to be useful in the treatment of chronic diseases
in
which glutamate dysfunction plays a role, such as chronic neurodegeneration,
Alzheimer's Disease, Huntington's Disease, Parkinson's Disease, amyotrophic
lateral
slerosis epilepsy, schizophrenia, AIDS dementia, or retinal diseases. Further,
compounds which inhibit or attenuate the release of glutamate may also provide
potential neuroprotective agents for the treatment of ischemia resulting from
stroke,
transient ischemic attack or brain//spinal trauma2 or of ischemia resulting
from
surgery where the blood flow must be halted for a period of time (for example,
cardiac by-pass surgery)3. Approximately 5-6 million people, in America alone,
are
afflicted with chronic or acute neurodegenerative disorders. Accordingly there
is a
need for an effective compound to treat and prevent neurodegenerative
conditions.
Therefore it is an object of the present invention to provide a method for the
treatment or prevention of neurodegenerative disorders.
It is another object of this invention to provide a source of neuroprotective
agents.
Further objects and features of the invention will become more apparent by
the detailed description set forth hereinbelow.
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Bibliography:
'Holt, W. F. et al, Glutamate in Health and Disease: The Role of Inhibitors,
Neuroprotection in CNS Diseases, Bar, P.R. and Beal, M.F., ed., Marcel Dekker,
Inc.,
New York, 1997, pp 87-199.
Engelsen, B.A. et al, Alterations in Excitatory Amino Acid Transmitters in
Human
Neurological Disease and Neuropathology, Neurotoxicity of Excitatory Amino
Acids,
Guidotti, A., ed., Raven Press Ltd., New York, 1990, pp. 311-332.
Ince, P.G., et al, The Role of Excitotoxicity in Neurological Disease, Res.
Contemp.
Pharmacother, (1997), 8, pp. 195-212.
Meldrum, B.S., The Gutamate Synapse as a Therapeutical Target: Perspective for
the Future, Prog. Brain Res., (1998),pp. 441-458.
2Koroshetz, W.J. andMoskowitz, M.A., Emerging Treatment for Stroke in Humans,
Trends in Pharmacol. Science, (1996), 17, pp. 227-233.
Dunn, C.D.R., Stroke: Trends, Tratments and Markets, Scrip Reports, PJB
Publications, Richmond Virginia, 1995.
3Arrowsmith, J.E., et al, Neuroprotection of the Brain During Cardiopulmonary
Bypass: A Randomized Trial of Remacemide During Coronary Artery Bypass in 171
Patients, Stroke, (1998), 29, pp. 2357-2362.
DESCRIPTION of DRAWINGS
Figure 1. Figure 1 is a schematic representation of the neuroprotective effect
of a 5-
HT6 agonist (Test Compound B) on neuronal survival, as determined by a
neurofilament ELISA.
Figure 2. Figure 2 is a schematic representation of the neuroprotective effect
of a 5-
HT6 agonist (Test Compound B) on neurite outgrowth wherein the data are
expressed as total neurite length.
Figure 3. Figure 3 is a schematic representation of the neuroprotective effect
of a 5-
HT6 agonist (Test Compound C) against OGD-induced neuronal cell death in
cerebellar granule neurons.
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Figure 4. Figure 4 is a schematic representation of the neuroprotective effect
of a 5-
HT6 agonist (Test Compound C) against Potassium withdrawal-induced apoptosis
in
cerebellar granule neurons.
Figure 5. Figure 5 is a schematic representation of the effect of a 5-HT6
agonist
(Test Compound B) on the brain derived neurotrophic factor (BDNF) protein
level in
cultured cortical neurons.
SUMMARY OF THE INVENTION
The present invention provides a method for the treatment of a
neurodegenerative disorder in a patient in need thereof which comprises
providing to
said patient a therapeutically effective amount of a 5-hydroxytryptamine-6
agonist.
Also provided is a pharmaceutical composition, for use in the treatment of a
neurodegenerative disorder, comprising a pharmaceutically acceptable carrier
and
an effective amount of a 5-hydroxytryptamine-6 agonist.
DETAILED DESCRIPTION OF THE INVENTION
Dysfunctional glutamate release, and in particular excessive glutamate
release, is associated with the pathohysiology of both acute neurodegenerative
disorders such as stroke, transient ischemic attack or spinal/brain trauma,
and
chronic neurodegenerative disorders such as epilepsy, Alzheimer's Disease,
amyotrophic lateral sclerosis, Huntington's disease, Parkinson's Disease, AIDS
dementia or retinal diseases.
Additionally, endogenous GABA function appears to be markedly decreased
in the brain following ischemic brain injury (Green A. R., et al.,
Neuroscience Letters,
1992, 138, 141-144; and Green A. R., et a/., Neuropharmacology, 2000, 39, 1483-
1493). Studies demonstrate that a drug capable of stimulating GABAergic
function
(eg., GABA agonist) when combined with an agent capable of decreasing
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glutamatergic neurotransmission (eg., glutamate antagonist) may have
neuroprotective effects (Lyden et al., Journal of Neurotrauma, 1995, 12(2),
223-230.
Moreover, brain derived neurotrophic factor (BDNF), a member of the nerve
growth factor family of proteins, has been shown to promote neuroprotection
and
neuroregeneration of neurons. (Binder, D. K. and Scharfman, H. E., Growth
Factors, 2004, 22(3), pp. 123-131) Compounds which increase the level of BDNF
may thereby promote the survival and plasticity of neurons and comensurately
demonstrate neuroprotective effects. (Nagappan, G. and Lu, B., Trends in
Neurosciences, 2005, 28(9), pp. 464-471).
Surprisingly, it has now been found that a 5-HT6 receptor agonist effectively
increases extracellular GABA concentrations and reduces glutamate release
caused
by ischemic-inducing agents. Further, it has now been found that a 5-HT6
agonist
effectively increases the level of brain derived neurotrophic factor (BDNF)
protein in
cultured cortical neurons. These findings strongly indicate that a 5-HT6
agonist has
neuroprotective properties, including promoting the survival and plasticity of
neurons,
and may be an effective therapeutic for the treatment and prevention of
neurodegenerative disorders.
Advantageously, the use of a selective 5-HT6 agonist for the treatment of
neurodegenerative disorders may have minimal side effects. Due to the
exclusive
localization of the 5-HT6 receptor in the brain, peripheral organ systems,
such as the
cardiovascular system, would not be affected by a 5-HT6 agonist. Further, the
specificity of the 5-HT6 agonist may lead to acute onset of action and
enhanced
therapeutic efficacy.
A 5-HT6 agonist is defined herein as any compound which is capable of
binding with the 5-HT6 receptor, as determined by conventional binding assay
methods well known in the art, and which demonstrates a 25% or greater,
preferably
50% or greater, more preferably 70% or greater, particularly 90% or greater,
accumulation of adenosine 3'5'-cyclic monophophate (cAMP) at the 5-HT6
receptor
site, as compared to serotonin.
Among the 5-HT6 agonists suitable for use in the method of the invention are
those compounds described in WO 99/47516, GB 2,341,549, US 6,770,642, US
6,767,912, US 6,800,640, US 6,727,246, and US 2003-0236278. US 6,770,642, US
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6,767,912, US 6,800,640, US 6,727,246, and US 2003-0236278 are incorporated
herein by reference thereto.
Methods to prepare 5-HT6 agonists suitable for use in the method of
invention are described in the above-mentioned patents and patent applications
and
also in US 4,940,710.
Preferred 5-HT6 agonists suitable for use in the method for the invention
include those compounds disclosed in WO 99/147516, GB 2,341,549, US 6,770,642
and US 2003-0236278 and having the structure of formula I
R, R5
R4
N
/R3
(I)
wherein
X is CH or N;
R, and R2 are each independently H, halqgen, CN, OC02R12, C02R13,
CONR14R15, CNR16NR17R,8, SOmR,9, NR20R21, OR22, COR23 or a C,-
C6alkyl, Cz-Cfialkenyl, Cz-Cfialkynyl, C3-C6cycloalkyl, cycloheteroalkyl,
aryl or heteroaryl group each optionally substituted;
R3 is S02R8 when X is CH or (CH2)nNR6R7 when X is N;
R4 is H, halogen, or a C,-Csalkyl, C,-Csalkoxy, aryl or heteroaryl group each
optionally substituted;
R5 is (CH2)nNR6R7 when X is CH or S02R8 when X is N;
n is an integer of 2 or 3;
R6 and R7 are each independently H or a C,-Csalkyl, C2-Csalkenyl, C2-
C6alkynyl, C3-C6cycloalkyl, cycloheteroalkyl, aryl or heteroaryl group
each optionally substituted, or R6 and R7 may be taken together with
the atom to which they are attached to form an optionally substituted
5- to 7-membered ring optionally containing an additional heteroatom
selected from 0, N or S;
R8 is an optionally substituted aryl, heteroaryl or 8- to 13-membered bicyclic
or tricyclic ring system having a N atom at the bridgehead and
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optionally containing 1, 2 or 3 additional heteroatoms selected from N,
O or S;
m is 0 or an integer of 1 or 2;
R12, R13, R19 and R23 are each independently H or a C,-Csalkyl, C2-C6alkenyl,
C2-Csalkynyl, C3-C6cycloalkyl, cycloheteroalkyl, aryl or heteroaryl
group each optionally substituted;
R14, R15 and R22 are each independently H or an optionally substituted C,-
C6alkyl group; and
R16, R17, R18, R20 and R21 are each independently H or an optionally
substituted C,-C4alkyl group; or R20 and R21 may be taken together
with the atom to which they are attached to form a 5- to 7-membered
ring optionally containing another heteroatom selected from 0, N or S;
or
the stereoisomers thereof or the pharmaceutically acceptable salts thereof.
As used in the specification and claims, the term halogen designates F, Cl, Br
or I and the term cycloheteroalkyl designates a 5-7 membered ring system
containing
1 or 2 heteroatoms, which may be the same or different, selected from
nitrogen,
oxygen and sulfur and optionally containing one double bond. Exemplary of the
cycloheteroalkyl ring systems included in the term as designated herein are
the
following rings wherein X, is NR, 0 or S; and R is H or an optional
substituent as
described hereinbelow:
_ \ N
xl X
X R x~ x
X1 1 1 1
-~ x' x
Xi X- X,J NJ NR
R
Similarly, as used in the specification and claims, the term heteroaryl
designates a five to ten membered aromatic ring system containing 1, 2 or 3
heteroatoms, which may be the same or different, selected from N, 0 or S. Such
heteroaryl ring systems include pyrrolyl, azolyl, oxazolyl, thiazolyl,
imidazolyl, furyl,
thienyl, quinolinyl, isoquinolinyl, indolinyl, benzothienyl, benzofuranyl,
benzisoxazolyl
or the like.
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The term aryl designates a carbocyclic aromatic ring system, e.g., having 6-
14 carbon atoms such as phenyl, naphthyl, anthracenyl or the like.
The term haloalkyl as used herein designates a C,H2n+, group having from
one to 2n+1 halogen atoms which may be the same or different and the term
haloalkoxy as used herein designates an OCnH2n+, group having from one to 2n+1
halogen atoms which may be the same or different.
Exemplary of the 8- to 13-membered bicyclic or tricyclic ring systems having a
N atom at the bridgehead and optionally containing 1, 2 or 3 additional
heteroatoms
selected from N, 0 or S included in the term as designated herein are the
following
ring systems wherein W2 is NR, 0 or S; and R is H or an optional substituent
as
described hereinbelow:
/ i i i YNl ~N N N N
I I I -
\ :N- NJ N Nv NJ \ NJ N N
~
N YN N" ~JN c~N Wz N
~NYNI ~ N YN N
I I I 1 1 ~ N
N_J N N~N-1 N C N
yd N i~ i i, W2~N1 N 1
~-f~ \ \ N- N 'WZ N
W2 N W2 N NY N, W2 N w2~ i
l; Y N, N-J J \' N \N '" N N
\ IN-~ Wz
WZ
/
Wz / N N i N \ ~N N ~ / N C(N N ~
N \ N
, L~J ~ N\ N
~'~ \
N N Wz~~ /~ ~~N'N
1 C\ J ~
~ \ N-N ~N-N ~N-N N
i N z / / N~
N
N ~ N.
_N!J N
~ ~ \ W2
In the specification and claims, when terms such as Cl-C6alkyl, C2-C6alkenyl,
C2-C6alkynyl, C3-C7cycloalkyl, cycloheteroalkyl, aryl or heteroaryl are
designated as
being optionally substituted, the substituent groups which are optionally
present may
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be one or more, e.g., 2 or 3, of those customarily employed in the development
of
pharmaceutical compounds or the modification of such compounds to influence
their
structure/activity, persistence, absorption, stability or other beneficial
property.
Specific examples of such substituents include halogen atoms, nitro, cyano,
hydroxyl,
alkyl, haloalkyl, alkoxy, haloalkoxy, amino, alkylamino, dialkylamino, formyl,
alkoxycarbonyl, carboxyl, alkanoyl, alkylthio, alkylsuphinyl, alkylsulphonyl,
carbamoyl,
alkylamido, phenyl, phenoxy, benzyl, benzyloxy, heterocyclyl (such as
heteroaryl or
cycloheteroalkyl) or cycloalkyl groups, preferably halogen atoms or lower
alkyl
groups. Typically, 0-3 substituents may be present. When any of the foregoing
substituents represents or contains an alkyl substituent as a group or part of
a group,
this may be linear or branched and may contain up to 12, preferably up to 6,
more
preferably up to 4 carbon atoms.
Compounds of formula I may be prepared according to the methods
described in GB 2,341,549, US 6,770,642 and US 2003-0236278.
More preferred 5-HT6 agonists suitable for use in the method of invention are
1-sulfonyltryptamine derivatives including those compounds of formula I
wherein X is
CH; n is 2; and R8 is a phenyl or imidazo[2,1-b][1,3]thiazolyl group each
optionally
substituted. Another group of more preferred 5-HT6 agonists suitable for use
in the
inventive method are 3-sulfonylazaindole derivatives including those compounds
of
formula I wherein X is N; n is 2; and R8 is a phenyl or imidazo[2,1-
b][1,3]thiazolyl
group each optionally substituted.
Among the 5-HT6 agonist compounds of formula I suitable for use in the
method of invention are: 2-{1-[6-chloroimidazo[2,1-b][1,3]thiazol-5-
yl)sulfonyl]-1H-
indol-3-yl}ethanamine; (2-{3-[(2,5-dimethoxyphenyl)sulfonyl]-1 H-pyrrolo[2,3-
b]pyridin-
1-yl}ethyl)amine; N-(2-{3-[(3-fluorophenyl)sulfonyl]-1 H-pyrrolo[2,3-b]pyridin-
l-
yl}ethyl)-N,N-dimethylamine; 2-{[1-(phenylsulfonyl)-1 H-indol-3-yl]ethyl}-N,N-
dimethylamine; the pharmaceutically acceptable salts thereof; or the
stereoisomers
thereof.
Compounds which exhibit 5-HT6 receptor agonist activity may form acid
addition salts with acids, such as conventional pharmaceutically acceptable
acids, for
example, acetic, phosphoric, sulfuric, hydrochloric, hydrobromic, citric,
maleic,
malonic, mandelic, succinic, fumaric, acetic, lactic, tartaric, salicylic,
nitric, sulfonic, p-
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toluene, sulfonic, methane sulfonic acid or the like. Salts of 5-HT6 receptor
agonists
are therefore embraced by the method of the invention.
The method of the invention includes esters, carbamates or other
conventional prodrug forms of a 5-HT6 agonist compound, which in general, are
functional derivatives of the 5-HT6 agonist compounds and which are readily
converted to the active moiety in vivo. Correspondingly, the method of the
invention
embraces the treatment of a neurodegenerative disorder with a 5-HT6 agonist,
such
as a compound of formula I or with a compound which is not specifically
disclosed
but which, upon administration, converts to a 5-HT6 agonist in vivo. Also
included
are metabolites of the 5-HT6 agonist compounds defined as active species
produced
upon introduction of said agonists into a biological system.
Compounds which exhibit 5-HT6 receptor agonist activity may exist as one or
more stereoisomers. The various stereoisomers include enantiomers,
diastereomers, atropisomers and geometric isomers. One skilled in the art will
appreciate that one stereoisomer may be more active or may exhibit beneficial
effects when enriched relative to the other stereoisomer(s) or when separated
from
the other stereoisomer(s). Additionally, the skilled artisan knows how to
separate,
enrich or selectively prepare said stereoisomers. Accordingly, the method of
invention embraces 5-HT6 agonist compounds, the stereoisomers thereof and the
pharmaceutically acceptable salts thereof. Said agonist compounds may be
present
as a mixture of stereoisomers, individual stereoisomers, or as an optically
active or
enantiomerically pure form.
Accordingly, the present invention provides an effective method for the
treatment and prevention of neurodegenerative disorders in a patient in need
thereof
which comprises providing to said patient a therapeutically effective amount
of a 5-
HT6 agonist as described hereinabove.
In one embodiment of the invention there is provided a method for increasing
brain-derived neurotrophic factor protein in a patient in need thereof which
comprises
providing to said patient a therapeutically effective amount of a 5-HT6
agonist as
described hereinabove
Said 5-HT6 agonist may be provided by oral or parenteral administration 'or by
any common manner known to be an effectual administration of a therapeutic
agent
to a patient in need thereof.
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A therapeutically effective amount, as used herein, is an amount sufficient to
provide a degree of neuroprotection, or to treat, prevent or ameliorate the
symptoms
associated with neurodegeneration or excessive or dysfunctional glutamate
release.
Neurodegenerative disorders suitable for treatment by the method of the
invention include both chronic neurodegenrative disorders and acute
neurodegenerative disorders. Chronic neurodegenerative disorders include, but
are
not limited to, Alzheimer's Disease, amyotrophic lateral sclerosis,
Huntington's
disease, Parkinson's Disease, AIDS dementia, epilepsy or retinal diseases.
Acute
neurodegenerative disorders include, but are not limited to, stroke, head or
spinal
trauma, or asphyxia. Stroke includes acute thromboembolic stroke, focal and
global
ischemia, transient cerebral ischemic attacks or other cerebral vascular
problems
accompanied by cerebral ischemia. Other acute neurodegenerative conditions are
associated with head trauma, spinal trauma, general anoxia, hypoxia, including
fetal
hypoxia, hypoglycemia, hypotension, as well as similar injuries seen during
procedures from embole, hyperfusion or hypoxia.
The method of invention may be useful in a range of incidents, including
during surgery, particularly cardiac surgery, in incidents of cranial
hemmorhage, in
perinatal asphyxia, in cardiac arrest, or status epilepticus, especially where
blood
flow to the brain is halted for a period of time.
The therapeutically effective amount provided in the treatment of a
neurodegenerative disorder may vary according to the size, age and response
pattern of the patient, the severity of the disorder, the judgment of the
attending
physician and the like. In general, effective amounts for daily oral
administration may
be about 0.01 to 1,000 mg/kg, preferably about 0.5 to 500 mg/kg and effective
amounts for parenteral administration may be about 0.1 to 100 mg/kg,
preferably
about 0.5 to 50 mg/kg.
In actual practice, said 5-HT6 agonist is provided by administering the 5-HT6
agonist compound or a precursor thereof in a solid or liquid form, either neat
or in
combination with one or more conventional pharmaceutical carriers or
excipients.
Accordingly, the present invention provides a pharmaceutical composition for
use in
the treatment and prevention of a neurodegenerative disorder which comprises a
pharmaceutically acceptable carrier and an effective amount of a 5-HT6 agonist
as
described hereinabove.
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Solid carriers suitable for use in the composition of the invention include
one
or more substances which may also act as flavoring agents, lubricants,
solubilizers,
suspending agents, fillers, glidants, compression aides, binders, tablet-
disintegrating
agents or encapsulating materials. In powders, the carrier may be a finely
divided
solid which is in admixture with a finely divided 5-HT6 agonist compound. In
tablets,
said 5-HT6 agonist compound may be mixed with a carrier having the necessary
compression properties in suitable proportions and compacted in the shape and
size
desired. Said powders and tablets may contain up to 99% by weight of the 5-HT6
agonist compound. Solid carriers suitable for use in the composition of the
invention
include calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin,
starch, gelatin, cellulose, methyl cellulose, sodium carboxymethyl cellulose,
polyvinylpyrrolidine, low melting waxes and ion exchange resins.
Any pharmaceutically acceptable liquid carrier suitable for preparing
solutions, suspensions, emulsions, syrups and elixirs may be employed in the
composition of the invention. The 5-HT6 agonist compound may be dissolved or
suspended in a pharmaceutically acceptable liquid carrier such as water, an
organic
solvent, or a pharmaceutically acceptable oil or fat, or a mixture thereof.
Said liquid
composition may contain other suitable pharmaceutical additives such as
solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring
agents,
suspending agents, thickening agents, coloring agents, viscosity regulators,
stabilizers, bsmo-regulators, or the like. Examples of liquid carriers
suitable for oral
and parenteral administration include water (particularly containing additives
as
above, e.g., cellulose derivatives, preferably sodium carboxymethyl cellulose
solution), alcohols (including monohydric alcohols and polyhydric alcohols,
e.g.,
glycols) or their derivatives, or oils (e.g., fractionated coconut oil and
arachis oil). For
parenteral administration the carrier may also be an oily ester such as ethyl
oleate or
isopropyl myristate.
Compositions of the invention which are sterile solutions or suspensions are
suitable for intramuscular, intraperitoneal or subcutaneous injection. Sterile
solutions
may also be administered intravenously. Inventive compositions suitable for
oral
administration may be in either liquid or solid composition form.
For a more clear understanding, and in order to illustrate the invention more
clearly, specific examples thereof are set forth hereinbelow. The following
examples
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are merely illustrative and are not to be understood as limiting the scope and
underlying principles of the invention in any way.
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EXAMPLE 1
Determination of the 5-HT6 Binding Affinity and cAMP Production of a Variety
of 5-HT6 Ligands
A) Evaluation of 5-HT6 Binding Affinity of Test Compounds
The affinity of test compounds for the serotonin 5-HT6 receptor is evaluated
in the following manner. Cultured Hela cells expressing human cloned 5-HT6
receptors are harvested and centrifuged at low speed (1,000 x g) for 10.0 min
to
remove the culture media. The harvested cells are suspended in half volume of
fresh
physiological phosphate buffered saline solution and recentrifuged at the same
speed. This operation is repeated. The collected cells are then homogenized in
ten
volumes of 50 mM Tris.HCI (pH 7.4) and 0.5 mM EDTA. The homogenate is
centrifuged at 40,000 x g for 30.0 min and the precipitate is collected. The
obtained
pellet is resuspended in 10 volumes of Tris.HCI buffer and recentrifuged at
the same
speed. The final pellet is suspended in a small volume of Tris.HCI buffer and
the
tissue protein content is determined in aliquots of 10-25 NI volumes. Bovine
Serum
Albumin is used as the standard in the protein determination according to the
method
described in Lowry et al., J. Biol. Chem., 193:265 (1951). The volume of the
suspended cell membranes is adjusted to give a tissue protein concentration of
1.0
mg/mI of suspension. The prepared membrane suspension (10 times concentrated)
is aliquoted in 1.0 ml volumes and stored at -70 C until used in subsequent
binding
experiments.
Binding experiments are performed in a 96 well microtiter plate format, in a
total volume of 200 pl. To each well is added the following mixture: 80.0 pl
of
incubation buffer made in 50 mM Tris.HCI buffer (pH 7.4) containing 10.0 mM
MgCI2
and 0.5 mM EDTA and 20 NI of [3H]-LSD (S.A., 86.0 Ci/mmol, available from
Amersham Life Science), 3.0 nM. The dissociation constant, Kp of the [3H]LSD
at the
human serotonin 5-HT6 receptor is 2.9 nM, as determined by saturation binding
with
increasing concentrations of [3H]LSD. The reaction is initiated by the final
addition of
100.0 NI of tissue suspension. Nonspecific binding is measured in the presence
of
10.0 pM methiothepin. The test compounds are added in 20.0 NI volume.
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The reaction is allowed to proceed in the dark for 120 min at room
temperature, at which time, the bound ligand-receptor complex is filtered off
on a 96
well unifilter with a Packard Filtermate 196 Harvester. The bound complex
caught
on the filter disk is allowed to air dry and the radioactivity is measured in
a Packard
TopCount equipped with six photomultiplier detectors, after the addition of
40.Opl
Microscinta-20 scintillant to each shallow well. The unifilter plate is heat-
sealed and
counted in a PackardTopCount with a tritium efficiency of 31.0%.
Specific binding to the 5-HT6 receptor is defined as the total radioactivity
bound less the amount bound in the presence of 10.OpM unlabeled methiothepin.
Binding in the presence of varying concentrations of test compound is
expressed as
a percentage of specific binding in the absence of test compound. The results
are
plotted as log % bound versus log concentration of test compound. Nonlinear
regression analysis of data points with a computer assisted program Prism
yielded
both the. IC50 and the K; values of test compounds with 95% confidence limits.
A
linear regression line of data points is plotted, from which the IC50 value is
determined and the K; value is determined based upon the following equation:
K; = IC50 / (1 + L/KD)
where L is the concentration of the radioactive ligand used and KD is the
dissociation
constant of the ligand for the receptor, both expressed in nM.
Using this assay, the following Ki values are determined and compared to
those values obtained by representative compounds known to demonstrate binding
to the 5-HT6 receptor. The data are shown in Table I below.
B) Determination of 5-HT6 Agonist Activity Using cAMP Accumulation
Intracellular cAMP levels are measured using 24-well plates containing the
human 5-HT6 receptor stabily transfected into HELA cells. Upon initiation of
the
assay, the media from cell maintenance is aspirated and cells are preincubated
at
37 C for 15 mins. in KREBS buffer. Following this primary incubation, the
buffer is
aspirated and an additional incubation is performed at 37 C for 5 mins. in
KREBS
buffer containing 500uM IBMX (3-isobutyl-l-methylxanthine). Subsequently cells
are
incubated with test compound concentrations ranging from 10-6 to 10-11 M for
10
minutes at 37 C. The assay is terminated by the addition of 0.5M perchloric
acid.
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Intracellular cAMP levels are determined by radioimmunoassay through the cAMP
SPA screening kit. Data are analyzed graphically with GraphPad Prism (GraphPad
Software, San Diego, CA). A 5-HT6 agonist is hereby defined as a compound
which
demonstrates >25% activity relative to the cAMP levels measured by the
addition of
serotonin (100nM). The value is recorded as Emax (%) and shown on Table I.
Table I
5-HT6
Test Compound Ki (nM) %Emax
A: (2-{3-[(2,5-dimethoxyphenyl)sulfonyl]-1 H- 3.6 95
pyrrolo[2,3-b]pyridin-1-yl}ethyl)amine
B: N-(2-{3-[(3-fluorophenyl)sulfonyl]-1 H-pyrrolo[2,3- 5.0 100
b]pyridin-1-yl}ethyl)-N, N-dimethylamine
C: 2-{1-[6-chloroimidazo[2,1-b][1,3]thiazol-5- 2.0 93
yl)sulfonyl]-1 H-indol-3-yl}ethanamine
EXAMPLE 2
Evaluation of a 5-HT6 Agonist in Neuronal Survival
In this evaluation neuron cultures are prepared from E16 rat embryos. After a
24h period, Test Compound B is added at various concentrations to the
cultures.
After a 72h period, neuronal survival is determined by a neurofilament ELISA.
The data are expressed as total neurofilament content. The EC50 for Test
Compound B in this evaluation is 50 nM. The results are shown in Figure 1
Results and Discussion:
As shown in figure 1, neuronal survival in culture is measured by the amount
of neurofilament present after 72 h. Treatment of cultured neurons with Test
Compound B increases the neurofilament content when compared to vehicle
treated
controls. The lowest concentration of Test Compound B providing significant
enhancement of survival is 10 nM.
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EXAMPLE 3
Evaluation of a 5-HT6 Agonist on Neurite Outgrowth in Cultured Cortical
Neurons
In this evaluation cortical neuron cultures are prepared from E16 rat embryos.
After a 24h period, Test Compound B is added at various concentrations to the
cultures. Neurite outgrowth is determined after a 72h period by staining cells
witha
tubulin antibody (TUJ-1) and measuring neurite length with the Cellomics
ArrayScan,
using the Enhanced Neurite Outgrowth (ENO) algorithm. The data are expressed
as
total neurite length. The EC50 for Test Compound B in this evaluation is 48
nM. The
results are shown in Figure 2
Results and Discussion:
As shown in figure 2, total neurite length of cultured neurons is quantified
after 72 h in culture. Treatment of cultured neurons with Test Compound B
increased total neurite length when compared to vehicle treated controls. The
lowest
concentration of Test Compound B providing significant enhancement of total
neurite
length is 10 nM.
EXAMPLE 4
Evaluation of the Neuroprotective Effect of a 5-HT6 Agonist Against Oxygen
and Glucose Deprivation in Cerebellar Granule Neurons
Preparation of Cerebellar Granule neurons (CGN):
Cerebella isolated from P7 rat pup brains are cut into 1 mm pieces and
transferred to a tube containing 0.3mg/ml trypsin in HBSS. Following enzymatic
digestion, the tissue is mechanically triturated. The supernatant is collected
and
centrifuged at 1200 rpm for 10 minutes. The resultant pellet is resuspended in
complete media (Neurobasal, 25nM potassium, 0.5mM L-glutamine, 100U/ml
penicillin, 100Ng/mI streptomycin, 10% FBS) and plated at a density of 0.5 x
106
cells/well in 24-well plates. After 24 h, media is exchanged with complete
serum-free
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medial (Neurobasal, 25nM potassium, 0.5mM L-glutamine, 100U/ml penicillin,
100Ng/mi streptomycin, B-27 supplement).
Oxygen glucose deprivation (OGD) in CGN:
This is a well characterized model of ischemia-like neuronal injury involving
glutamate-induced excitotoxicity (A. Kaasik, et al, Neuroscience (2001) 102,
pp427-
432). Cultures are maintained for 14 days in vitro prior to experimentation.
Cultures
are pretreated for 1 h with various concentrations of Test Compound C and
transferred to an anaerobic chamber, where the media is exchanged with
deoxygenated buffer and maintained for 4 h in the presence of fresh Test
Compound
C. Following the 4 h OGD, deoxygenated buffer is exchanged with complete
media.
Cultures are maintained for 24 h in the presence of fresh Test Compound C. At
the
end of the 24-h period, cell death is determined by measuring lactate
dehydrogenase
(LDH) released into the media and is expressed as a percent of cell death. The
results are shown in Figure 3 and are presented as means SD from 5
experiments.
Sham bar represents results obtained with cultures subjected to manipulations
and
changes of culture media without OGD or drug treatment.
Results and Discussion:
As shown in figure 3, exposure of CGN cultures to 4 h of OGD results in over
50% of cell death as measured by a release of a cytoplasmic enzyme (LDH) into
the
culture medium. Pretreatment of cultured neurons with Test Compound C,
followed
by their exposure to OGD and a 24 h recovery period in the presence of Test
Compound C results in a dose-dependent reduction of neuronal cell death. The
lowest concentration of Test Compound C providing significant protection is
10nM.
Treatment with 1 pM Test Compound C reduced neuronal death by 50%.
EXAMPLE 5
Evaluation of the Neuroprotective Effect of a 5-HT6 Agonist Against Potassium
Withdrawal-Induced Apoptosis in Cerebellar Granule Neurons
Potassium withdrawal, i.e. replacement of 25nM potassium in the CGN
culture medium is a well established model of neuronal cell death by apoptosis
(T. M.
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Miller and E. M. Johnson, Journal of Neuroscience, (1996) 16(23), pp7487-
7495).
Cultures are maintained for 7 days prior to experimentation. Complete media
containing 25mM K+ is exchanged with complete media containing 5mM K. Various
concentrations of Test Compound C are added to the cultures. After 24 h,
apoptotic
cell death is determined by measuring DNA fragmentation via ELISA. Apoptotic
cell
death is expressed as % apoptosis. The results are presented as means SD
from
two experiments and are shown in Figure 4.
Results and Discussion:
As can be seen in Figure 4, a replacement of 25mM potassium with 5mM
potassium induces 75% apoptotic neuronal cell death after a 24 h interval. The
presence of Test Compound C during the low potassium treatment significantly,
and
dose dependently, attenuated the amount of fragmented DNA used as a measure of
apootosis in this model. The lowest concentration of Test Compound C evaluated
(0.3pM) reduced cell death by 50% while almost total protection from apoptosis
was
found in the presence of 3.OpM of Test Compound C.
EXAMPLE 6
Evaluation of the Effect of a 5-HT6 Agonist on Brain Derived Neurotrophic
Factor Protein Levels in Cultured Cortical Neurons
In this evaluation cortical neuron cultures are prepared from E16 rat embryos
and plated on pre-coated 10 cm Petri dishes. After a 24h period, Test Compound
B
is added to the cultures. Levels of brain derived neurotrophic factor (BDNF)
protein
were measured after a 72h period by lysing the cells and using a BDNF sandwich
ELISA. More specifically, ELISA plates were coated with an anti-BDNF
monoclonal
antibody. Nonspecific binding was blocked and 50 g of sample protein was
added.
A second anti-BDNF antibody was added and incubated. An anti-IgY antibody
conjugated with horseradish peroxidase was added and incubated. A TMB solution
was added and the colorimetric reaction was measured in a plate reader
(absorbance of 450 nm). BDNF levels are quantified after 72 h in culture. The
data
are graphically shown in Figure 6.
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Results and Discussion:
As shown in figure 6, treatment of cultured neurons with Test Compound B
significantly increased BDNF protein levels when compared to vehicle treated
control.
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